The present invention relates to a diagnostic drug for and a kit for diagnosing autoimmune diseases, and a method for detecting an antibody of an autoimmune disease patient using high mobility group protein-1 (HMG-1), high mobility group protein-2 (HMG-2), or a fragment of a polypeptide thereof with which the antibody of the autoimmune disease patient reacts. In particular, the present invention relates to a diagnostic drug for and a kit for diagnosing rheumatoid arthritis, systemic lupus erythematosus, Sjxc3x6gren""s syndrome, Behxc3xa7et""s disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis/polyarteritis nodosa, ulcerative colitis, Crohn""s disease, and autoimmune hepatitis, and a method for detecting an antibody of a patient of any of the above-mentioned diseases, using HMG-1, HMG-2, or a fragment of a polypeptide thereof with which the antibody of such a patient reacts.
It has been reported that various anti-neutrophil cytoplasmic antibodies (ANCA) are involved in autoimmune and inflammatory diseases. ANCA are antibodies capable of being detected by indirect immunofluorescence assay (IIF) and are classified into cytoplasmic-ANCA (cANCA) and perinuclear-ANCA (pANCA). cANCA is detected in Wegener""s granulomatosis patients at a frequency of as high as 80%, and the antigen to cANCA is proteinase-3 (PR-3) in 90% or more of all the cases. pANCA is detected in microscopic polyangitis, and pauci-immune necrotizing crescentic glomerulonephritis (NCGN) patients at a frequency of as high as 80%, and the antigen to the antibody is myeloperoxidase (MPO-ANCA) in 80% of all the cases. Early-phase diagnosis and differential diagnosis of angiitis syndromes are realized by measurement of highly disease-specific antibodies.
Recently, pANCA has been found in the patients suffering from inflammatory diseases including inflammatory bowel disease (IBD) such as ulcerative colitis (UC), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), autoimmune hepatitis (AIH), malignant tumors, amebic abscesses, and sweet disease. As antigens to pANCA, lactoferrin, cathepsin G, elastase, lysozyme and the like have been identified. Causes of these diseases and relationship of these antigens and the diseases have been studied. The specificity of these antigens to pANCA is low, which suggests that there are other antigens involved.
Generally, the ratios at which ANCA is detected in the patients (percentages of positive patients) of ulcerative colitis (UC) and Crohn""s disease (CD), which are both inflammatory bowel diseases, by the indirect immunofluorescence assay are 40 to 87% and 6 to 27%, respectively. Judging by the staining pattern, pANCA is detected at a high percentage of 80 to 95% in the case of ulcerative colitis, whereas pANCA and cANCA are equivalently detected in the case of Crohn""s disease. pANCA is detected at a high percentage in the case of rheumatoid arthritis, systemic lupus erythematosus and Sjxc3x6gren""s syndrome, and the percentages are 33% (4%), 43% (2%) and 50% (8%), respectively. The numerals in parentheses are percentage of cANCA-positive patients.
As antigens to ANCA detected in ulcerative colitis and Crohn""s disease, various antigens have been reported including lactoferrin, cathepsin G, myeloperoxidase, and myeloperoxidase+elastase, and myeloperoxidase+elastase+cathepsin G. However, antigens specific to these diseases have not been identified so far. Antigens of other diseases involving pANCA have not been identified, either.
Standard diagnostic methods for ulcerative colitis and Crohn""s disease include endoscopy, biopsy, and X-ray examination. These methods are costly, painful, and time-consuming. Recently, detection of pANCA by indirect immunofluorescence assay has been reported as a serodiagnosis for ulcerative colitis. However, this method is not sufficiently sensitive and tends to have an increased background signal. Serodiagnosis, by which neutrophils and other cells are fixed on a plate with ethanol, has a further disadvantage in that the result depends on the state of the cells and the fixing technique and is not sufficiently reliable. Accordingly, serodiagnosis has not been in general use. Regarding Crohn""s disease, even an autoantibody has not been found. As described above, a specific and simple diagnostic method for autoimmune diseases has not been developed.
In rheumatoid arthritis and systemic lupus erythematosus, various autoantibodies such as a rheumatoid factor and antinuclear antibody are produced. In scleroderma, polymyositis, Behxc3xa7et""s disease, and periarteritis nodosa, few autoantibodies are detected and thus clinical symptoms are main factors for diagnosis. It has been pointed out that early diagnosis of these diseases is important to prevent progress of the disease in prognostic phase by sufficient therapy. However, a simple serodiagnostic method has not been developed.
In general, when a patient is suspected to have an autoimmune disease, examination of an antinuclear antibody is used for diagnosis at primary screening. Antinuclear antibody is a general term for autoantibodies, the antigens to which are cell nucleic acids and various nucleic protein components, and includes various antibodies. Antinuclear antibodies are mainly detected by the indirect immunofluorescence assay. As the antinuclear antibodies, anti-DNA antibody, anti-histone antibody, anti-ENA antibody, anti-centromere antibody, and anti-nuclear antibody are assumed which are classified by the staining pattern. However, it has been pointed out that there are many problems in uniformalizing the measurement precision of antinuclear antibodies. The problems include, for example, that the nucleus materials (cells) vary in accordance with the research facilities, the calibration curve cannot be obtained, and the autoantibodies are non-uniform. The most serious problem is that the method relies on visual observation and thus the determination criterion and technology are not objective.
Despite these problems, measurement of antinuclear antibodies by indirect immunofluorescence assay is now indispensable to diagnosis of various collagen diseases including systemic lupus erythematosus and understanding of clinical conditions thereat. Needless to say, it is demanded to identify autoantigens of autoimmune diseases other than the antinuclear antibody and to develop a simple and objective method for detecting antibodies using the antigens, with no difference among research facilities.
As can be appreciated, it leads to establishment of diagnosis of an autoimmune disease and appropriate therapeutic strategies to identify an autoantigen of an autoimmune disease patient and to detect an autoantibody using the autoantigen. It is demanded to identify and isolate a common autoantigen in autoimmune diseases and to develop a simple method for detecting an antibody using the antigen.
As a result of active studies in order to overcome the above-described problems with the prior art, the present inventors succeeded for the first time in history in isolating high mobility group protein-1 (HMG-1) and high mobility group protein-2 (HMG-2), which are known proteins, using antibodies in the sera of the autoimmune disease patients, especially pANCA-positive ulcerative colitis patients. HMG-1 and HMG-2 were isolated and identified as novel antigens with which the antibodies react (Sobajima J. et al., Clin. Exp. Immunol. 105:120-124, 1996; Sobajima J. et al., Clin. Exp. Immunol. 107:135-140, 1997). By constructing an ELISA system using the HMG-1 and HMG-2, the present inventors showed that a high percentage of antibodies of the patients of rheumatoid arthritis, systemic lupus erythematosus, Sjxc3x6gren""s syndrome, Behxc3xa7et""s disease, scleroderma, primary biliary cirrhosis, microscopic polyanglitis/polyarteritis nodosa, ulcerative colitis, Crohn""s disease and autoimmune hepatitis are positive to these antigens. By showing that the ELISA system is more sensitive, simpler, more reliable and more objective than measurement for antinuclear antibodies by indirect immunofluorescence assay, the present inventors found that antibodies against the antigens can be markers for diagnosis of rheumatoid arthritis, systemic lupus erythematosus, Sjxc3x6gren""s syndrome, Behxc3xa7et""s disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis/polyarteritis nodosa, ulcerative colitis, Crohn""s disease and autoimmune hepatitis. Thus, the present invention has been completed.
The HMG antigens were previously measured as one of antinuclear antibodies of autoimmune diseases, not as an antigen to ANCA. Dennis J. S. et al. reported at the systemic lupus erythematosus patients have an anti-HMG-1 antibody at a radio of 10. % and an anti-HMG-2 antibody at a ratio of 6.9%, and that the mixed connective-tissue disease patients and the rheumatoid arthritis patients have both antibodies at a ratio of 0% (Science 215, 1245-1247, 1982). Briolay J. et al. detected an anti-HMG-1 antibody and an anti-HMG -2 antibody by immunoblotting assay from the patients of systemic lupus erythematosus, rheumatoid arthritis and scleroderma, and reported that neither antibody has a diagnostic value (Autoimmunity, 2, 165-176, 1989). It is assumed they had problems in the purity of the HMG antigens, the techniques such as the ELISA assay and the immunoblotting assay, the state of e patients and the number of the subjects, but an invention relating to a diagnostic drug using 11MG-1 and HMG-2 had not been completed at the time of the above-mentioned publications. There is a report that 39% of the antinuclear antibody-positive juvenile rheumatism patients are positive with respect to the anti-HMG-1 antibody and/or anti-HMG-2 antibody (Wittemann B. et at., Arthritis and Rheumatism 33, 1378-1383, 1990). The present inventors constructed as ELISA system using highly pure HMG-1 and HMG-2 and measured the percentage of the patients who were positive to these antigens regarding various diseases. As a result, a significant difference from the healthy (or normal) persons was found in ten diseases. Thus, the present invention has been completed.
The present invention relates to a diagnostic drug for autoimmune diseases, comprising at least one of a polypeptide selected from an HMG-1 family, a polypeptide selected from an HMG-2 family, a fragment thereof which is reactable with an antibody of an autoimmune disease patient, a kit for diagnosing these diseases, and a method for detecting an antibody of an autoimmune disease patient. When the polypeptide is a neutrophil 28 kDa antigen (HMG-2), the autoimmune disease is not ulcerative colitis.
In a preferred embodiment of the invention, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, Sjxc3x6gren""s syndrome, Behxc3xa7et""s disease, scleroderma, primary biliary cirrhosis, microscopic polyangiitis/polyarteritis nodosa, ulcerative colitis, Crohn""s disease, or autoimmune hepatitis.
In a preferred embodiment of the invention, the polypeptide is selected from human, bovine, porcine, chicken, mouse or rat HMG-1 or HMG-2.
Thus, the objectives of the present invention are achieved.